Information on EC 3.4.22.49 - separase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
3.4.22.49
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RECOMMENDED NAME
GeneOntology No.
separase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
all bonds known to be hydrolysed by this endopeptidase have arginine in P1 and an acidic residue in P4. P6 is often occupied by an acidic residue or by an hydroxy-amino-acid residue, the phosphorylation of which enhances cleavage
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
CAS REGISTRY NUMBER
COMMENTARY hide
351527-77-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
several strains
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Manually annotated by BRENDA team
gene ESP1
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Manually annotated by BRENDA team
gene Separase
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
gene Os02g0770700
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Manually annotated by BRENDA team
gene Separase
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Manually annotated by BRENDA team
strain SK1
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Manually annotated by BRENDA team
gene XM_002454579
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
gene LOC100259948
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
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Plk1-mediated phosphorylation of Cdc6 on residue T37 promotes the interaction of Cdc6 and Cdk1, leading to the attenuation of Cdk1 activity, release of separase, and subsequent anaphase progression
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acetyl-Asp-Arg-Glu-Ile-Nle-Arg-7-amido-4-methylcoumarin + H2O
acetyl-Asp-Arg-Glu-Ile-Nle-Arg + 7-amino-4-methylcoumarin
show the reaction diagram
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acetyl-Asp-Arg-Glu-Ile-Nle-Arg is the Rad21-peptidyl
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-
?
Cdc14 + H2O
?
show the reaction diagram
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separase regulates INCENP-Aurora B anaphase spindle function through activation of Cdc14
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-
?
cohesin + H2O
?
show the reaction diagram
cohesin + H2O
cleaved cohesin
show the reaction diagram
cohesin + H2O
fragments of cohesin
show the reaction diagram
kendrin + H2O
?
show the reaction diagram
Rad 21 + H2O
?
show the reaction diagram
-
-
-
-
?
Rad21 + H2O
cleaved Rad21
show the reaction diagram
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Rad21 is cleaved by Cut1 at the metaphase-anaphase transition
-
-
-
Rad21 + H2O
fragments of Rad21
show the reaction diagram
Rec8 + H2O
?
show the reaction diagram
Rec8 + H2O
cleaved Rac8
show the reaction diagram
Rec8 + H2O
fragments of Rac8
show the reaction diagram
Rec8 + H2O
fragments of Rec8
show the reaction diagram
Scc1 + H2O
?
show the reaction diagram
Scc1 + H2O
cleaved Scc1
show the reaction diagram
Scc1 + H2O
fragments of Scc1
show the reaction diagram
Slk19 + H2O
?
show the reaction diagram
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a protein implicated in the mitotic exit via its role in the stabilization of spindle in budding yeast
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-
?
Slk19 + H2O
cleaved Slk19
show the reaction diagram
Slk19 + H2O
fragments of Slk19
show the reaction diagram
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separase cleaves the kinetochore-associated protein Slk19 in anaphase
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?
SYN1 + H2O
?
show the reaction diagram
the enzyme plays an essential role in embryo development. The enzyme is required for the removal of cohesin from meiotic chromosomes. The cleavage of SYN1 by separase is responsible for the release of sister chromatid cohesion during meiosis
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-
?
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
Cdc14 + H2O
?
show the reaction diagram
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separase regulates INCENP-Aurora B anaphase spindle function through activation of Cdc14
-
-
?
cohesin + H2O
?
show the reaction diagram
cohesin + H2O
cleaved cohesin
show the reaction diagram
cohesin + H2O
fragments of cohesin
show the reaction diagram
kendrin + H2O
?
show the reaction diagram
Rad 21 + H2O
?
show the reaction diagram
-
-
-
-
?
Rad21 + H2O
cleaved Rad21
show the reaction diagram
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Rad21 is cleaved by Cut1 at the metaphase-anaphase transition
-
-
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Rad21 + H2O
fragments of Rad21
show the reaction diagram
-
-
-
-
?
Rec8 + H2O
?
show the reaction diagram
Rec8 + H2O
cleaved Rac8
show the reaction diagram
Rec8 + H2O
fragments of Rec8
show the reaction diagram
Scc1 + H2O
?
show the reaction diagram
Scc1 + H2O
cleaved Scc1
show the reaction diagram
Scc1 + H2O
fragments of Scc1
show the reaction diagram
-
Scc1 is a subunit of cohesion, centrosomal Scc1 is cleaved by separase coincidentally with chromatin Scc1
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-
?
Slk19 + H2O
?
show the reaction diagram
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a protein implicated in the mitotic exit via its role in the stabilization of spindle in budding yeast
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-
?
Slk19 + H2O
cleaved Slk19
show the reaction diagram
SYN1 + H2O
?
show the reaction diagram
Q5IBC5
the enzyme plays an essential role in embryo development. The enzyme is required for the removal of cohesin from meiotic chromosomes. The cleavage of SYN1 by separase is responsible for the release of sister chromatid cohesion during meiosis
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-
?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Aki1
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-
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astrin
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Cdc55
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results suggest that Cdc55 acts as inhibitor downstream from shugoshin
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Cdk1 kinase
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phosphorylation-dependent binding of the cyclin B1 subunit of Cdk1 kinase to a Cdc6-like domain of separase inhibits separase
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cyclin B
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cyclin B1
cyclin-dependent kinase 1-cyclin B1
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Cdk1-cyclin B1 triggers precipitation of separase by phosphorylation but stabilizes it by inhibitory binding. Only separase that is first complexed by Cdk1-cyclin B1 can later be activated by cyclin B1 degradation
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Pds1
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Pds1 is a chaperone, inhibition of Esp1 by overexpression of undegradable Pds1 blocks mitotic exit via blockage of cohesin cleavage
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peptide
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acyloxymethyl ketone derivative of human SCC1 cleavage site peptide, chloromethyl ketone derivatives of the yeast Scc1 cleavage site
securin
securin Pds1
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an inhibitor of separase Esp1 in budding yeast. As Pds1 is degraded, Esp1 is activated, and cells transit into anaphase
securing
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the motif containing H134 in securing isoform PTTG1 has a strong affinity for separase and is involved in inhibiting it, while another domain(s) in isoform is involved in activating separase and has a weaker affinity for it. Isoform PTTG2 does not interact with separase
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sercurin
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securin persistently binds and inhibits separase during much of metaphase
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shugoshin
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prevents separase activation independently of securin, protein phosphatase 2A coupled to regulatory subunit Cdc55 is essential for Shugoshin-mediated inhibition
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additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cell division cycle 6
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Cdc6, a mitotic substrate of polo-like kinase. Cleavage of chesin/Rad21 is much greater in wild-type Cdc6 expressing cells than in GFP and Cdc6-T37V mutant expressing cells
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cyclinB1
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imatinib
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activation of separase proteolytic activity occurs exclusively in BCR-ABL-positive cells during imatinib treatment (0.00025-0.01 mM for 24 h)
PP2A
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securin
securing
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.293
acetyl-Asp-Arg-Glu-Ile-Nle-Arg-7-amido-4-methylcoumarin
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at 37C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 55
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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in a murine model of absent securin expression, the PTTG knock-out mouse, separase and Rad21 are over-expressed in multiple brain regions. Furthermore, Rad21 mRNA expression is highly correlated with that of securin, separase, cyclin C and sestrin 2 in fetal brains
Manually annotated by BRENDA team
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highly expressed in human fetal cerebral cortex compared with adult
Manually annotated by BRENDA team
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separase expression is very high in both normal nonneoplastic and neoplastic colons
Manually annotated by BRENDA team
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separase localizes to the ingressing furrow and midbody during cytokinesis in the Caenorhabditis elegans embryo
Manually annotated by BRENDA team
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diploid FSK3 mouse mammary epithelial cells
Manually annotated by BRENDA team
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increased expression of separase
Manually annotated by BRENDA team
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increased expression of separase
Manually annotated by BRENDA team
additional information
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human separase is present in cells as a part of very large protein complex, which in addition to securin contains also Cdk and cyclin B1, both able to inhibit separase
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
126000
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calculated mass of the native separase, SDS-PAGE, Western blot
160000
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autocleavage fragment of separase, SDS-PAGE
220000
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full-length separase, SDS-PAGE
225000
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recombinant separase, immunoblotting
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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the human separase is composed of three domains: the tail, the trunk, and the head, structure modeling. The first two domains are spanned by Armadillo, ARM, repeats, which are composed of multiple 42 amino acid repeats and are present in the proteomes of all eukaryotic organisms. The ARM repeat domain is highly conserved right-handed super helix of ?-helices, which serves as molecular scaffold for protein-protein interactions. Phosphorylation and potential autocleavage sites span the region of the last ARM repeats and the central unstructured region. Human separase has an N-terminal region spanned by 26 ARM repeats and separated from the
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
proteolytic modification
additional information
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phosphorylation and potential autocleavage sites span the region of the last ARM repeats and the central unstructured region. Human separase has an N-terminal region spanned by 26 ARM repeats and separated from the two caspase-like domains, one of which is active, by the unstructured region
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
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no appreciable activity is observed at 4C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Cdc48 is required for the stability of Cut1/separase in mitotic anaphase
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, 1 week, no significant loss of activity
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23C, 48 h, 17% loss of activity
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4C, 48 h, 12% loss of activity
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temperature, storage medium, duration, loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
separase is expressed as a GST- or myc-tagged fusion protein in Escherichia coli BL-21 and HEK-293 cells
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
analysis of correlation between overexpression of separase and aneuploidy in tetracycline-inducible diploid FSK3 mouse mammary epithelial cells
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continuous overexpression of cmyc tagged TbSep is lethal to the cells
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depletion of endogenous separase with siRNA in NSCLCs shows that separase affects progression through the G2 phase of mitosis. Analysis of the effect of separase depletion on irradiated cells (cell cycle progression, M-phase fraction, late mitotic-linked cell death and clonogenic survival)
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enhanced cyclin-dependent kinase 1 activity due to deregulation of CDC27-anaphase-promoting complex leads in turn to hyperphosphorylation of Separase, impeding chromatid separation. Regulation of mitotic progression by transforming growth factor-beta in bone marrow stromal cells is through targeting the APC-separase pathway
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expressed in HEK-293T cells
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expressed in HeLa cells
expression of myc6-separase in 293T cells
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expression of wild-type and C2029 mutant enzyme in 293 EcR and HeLa cells
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generation of separase gene knock-out mutants
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investigation of the expression level of separase across a wide range of human tumors. Overexpression of separase transcripts strongly correlates with high incidence of relapse, metastasis and lower 5-year overall survival rate in breast and prostate cancer patients.
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mutations introducing substitutions throughout the Esp1 polypeptide prevent loss of sister chromatid cohesion and cause mitotic failure. Generation of esp1-temperature sensitive mutant for functional analysis of further enzymes
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the active (S1126A/T1346A) and the inactive (C1129S) separase mutants are coexpressed in HEK-293T cells with human securin
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Zds1 is required for Separase-induced Cdc14 activation. Analysing the role of proteins Zds1 and Zds2 in mitotic exit machinery
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
decreased separase protein levels are observed in BCR-ABL-positive and -negative cells treated with imatinib in a dose dependent manner
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separase is overexpressed and mislocalized in a wide range of human cancers, including breast, prostate, and osteosarcoma
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C1129S
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inactive
C2029A
E1483R/R1486E
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autoproteolytically cleaved like wild-type
E1503R/R1506E
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autoproteolytically cleaved like wild-type
E1532R/R1535E
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autoproteolytically cleaved like wild-type
R1486A
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mutation fails to block autocatalytical cleavage
R1506A
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mutation at autocatalytical cleavage site, mutant enzyme is still cleaved indicating the existence of a second cleavage site
R1506A/R1486A
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mutation fails to block autocatalytical cleavage
S1126A
S1126A/T1346A
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active mutant
T1346A/T1363A/S1399A
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this triple mutant binds relatively more Cdk1 than S1126A but its Cdk1 binding capacity is much decreased compared to wildtype. A doxycycline-induced overexpression of the triple mutant in HEK-293 cells leads to an increase of the G2/M cell population from 24 to 37% within 24 h compared to wild-type. Giemsa-stained prometaphase-chromosomes from triple mutant expressing cells are mostly separated (61%), whereas those from wild-type or T1346E/T1363E/S1399D expressing cells are almost always paired (78 and 83%, respectively)
T1346A/T1363A/S1399D
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the mitotic arrest phenotype caused by the T1346A/T1363A/S1399A triple mutant is largely abrogated when just residue 1399 is changed to aspartate. Thus among the phosphorylation sites within the Cdc6-like domain serine 1399 is sufficient to support Cdk1-dependent inhibition of separase
T1346E/T1363E/S1399D
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changing the phosphorylation sites within the Cdc6-like domain to acidic residues results in a separase that can still be inhibited by Cdk1 in vivo
T1346E/T1363E/S1399S
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the mitotic arrest phenotype caused by the T1346A/T1363A/S1399A triple mutant is largely abrogated when just residue 1399 is changed to serine. Thus among the phosphorylation sites within the Cdc6-like domain serine 1399 is sufficient to support Cdk1-dependent inhibition of separase
S1121A
S1126A
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specifically abolished separase hyperphosphorylation in Smad3-deficient cells
H1531A
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active site point mutation prevents Scc1 from being cleaved after binding
S1138A
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mutant containing phosphorylation site mutation compromises Cdk1-inhibitory ability
S1139A
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mutant containing phosphorylation site mutation compromises Cdk1-inhibitory ability
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
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the results show that separase might be a tumor supressor gene
molecular biology